Changes in the patterns of gene expression are widely believed to underlie many of the phenotypic differences within and between species. Although much emphasis has been placed on changes in transcriptional regulation, gene expression is regulated at many levels, all of which must ultimately be studied together to obtain a complete picture of the evolution of gene expression. Here we compare the evolution of transcriptional regulation and post-transcriptional regulation that is mediated by microRNAs, a large class of small, non-coding RNAs in plants and animals, focusing on the evolution of the individual regulators and their binding sites. As an initial step towards integrating these mechanisms into a unified framework, we propose a simple model that describes the transcriptional regulation of new microRNA genes

MicroRNAs (miRNAs) are emerging as important, albeit poorly characterized, regulators of biological processes. Key to further elucidation of their roles is the generation of more complete lists of their numbers and expression changes in different cell states. Here, we report a new method for surveying the expression of small RNAs, including microRNAs, using Illumina sequencing technology. We also present a set of methods for annotating sequences deriving from known miRNAs, identifying variability in mature miRNA sequences, and identifying sequences belonging to previously unidentified miRNA genes. Application of this approach to RNA from human embryonic stem cells obtained before and after their differentiation into embryoid bodies revealed the sequences and expression levels of 334 known plus 104 novel miRNA genes. One hundred seventy-one known and 23 novel microRNA sequences exhibited significant expression differences between these two developmental states. Owing to the increased number of sequence reads, these libraries represent the deepest miRNA sampling to date, spanning nearly six orders of magnitude of expression. The predicted targets of those miRNAs enriched in either sample shared common features. Included among the high-ranked predicted gene targets are those implicated in differentiation, cell cycle control, programmed cell death, and transcriptional regulation.

MicroRNAs (miRNAs) are short endogenous RNAs known to post-transcriptionally repress gene expression in animals and plants. A microarray profiling survey revealed the expression patterns of 175 human miRNAs across 24 different human organs. Our results show that proximal pairs of miRNAs are generally coexpressed. In addition, an abrupt transition in the correlation between pairs of expressed miRNAs occurs at a distance of 50 kb, implying that miRNAs separated by <50 kb typically derive from a common transcript. Some microRNAs are within the introns of host genes. Intronic miRNAs are usually coordinately expressed with their host gene mRNA, implying that they also generally derive from a common transcript, and that in situ analyses of host gene expression can be used to probe the spatial and temporal localization of intronic miRNAs.

The majority of human microRNA (miRNA) loci are located within intronic regions and are transcribed by RNA polymerase II as part of their hosting transcription units. The primary transcripts are cleaved by Drosha to release B70 nt pre-miRNAs that are subsequently processed by Dicer to generate mature B22 nt miRNAs. It is generally believed that intronic miRNAs are released by Drosha from excised introns after the splicing reaction has occurred. However, our database searches and experiments indicate that intronic miRNAs can be processed from unspliced intronic regions before splicing catalysis. Intriguingly, cleavage of an intron by Drosha does not significantly affect the production of mature mRNA, suggesting that a continuous intron may not be required for splicing and that the exons may be tethered to each other. Hence, Drosha may cleave intronic miRNAs between the splicing commitment step and the excision step, thereby ensuring both miRNA biogenesis and protein synthesis from a single primary transcript. Our study provides a novel example of eukaryotic gene organization and RNAprocessing control

Many of microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) are located within the introns of genes in eukaryotes. Contrary to intronic snoRNAs, intronic miRNAs are processed from unspliced intronic regions before the catalysis of splicing in vertebrates. By analyzing the distribution patterns of the length and position of the introns hosting these two groups of small RNA genes, we observed that both human and mouse intronic miRNAs tended to be present in large introns, and miRNA host introns have a more 5′-biased position distribution compared with all other introns among the two genomes. These observations indicate that the negative selection of functional constraints might affect the intron size in both genomes. Interestingly, the very 5′-biased positions of miRNA host introns may be necessary for the transcription and regulation of intronic miRNAs to utilize the regulatory signals within the 5′-UTRs of their host genes.

MicroRNAs are short, noncoding RNAs that play important roles in post-transcriptional gene regulation. Although many functions of microRNAs in plants and animals have been revealed in recent years, the transcriptional mechanism of microRNA genes is not well-understood. To elucidate the transcriptional regulation of microRNA genes, we study and characterize, in a genome scale, the promoters of intergenic microRNA genes in Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, and Oryza sativa. We show that most known microRNA genes in these four species have the same type of promoters as protein-coding genes have. To further characterize the promoters of microRNA genes, we developed a novel promoter prediction method, called common query voting (CoVote), which is more effective than available promoter prediction methods. Using this new method, we identify putative core promoters of most known microRNA genes in the four model species. Moreover, we characterize the promoters of microRNA genes in these four species. We discover many significant, characteristic sequence motifs in these core promoters, several of which match or resemble the known cis-acting elements for transcription initiation. Among these motifs, some are conserved across different species while some are specific to microRNA genes of individual species.

Identification of microRNAs (miRNAs) is essential to studying their physiological functions. Due to the difficulties in discovering truly expressed miRNAs from genomic random hairpin secondary structure sequences, it is beneficial to predict them from expressed sequences—expressed sequence tags (ESTs) and intronic sequences. We used a modified scanning pipeline using criteria based on the features of known pre-miRNAs and phylogenetic conservation for predicting intronic miRNAs. Upon examination, we found that 25% of known human miRNAs belong to intronic regions of known protein-coding genes. About 50% of these intronic miRNAs reside in introns whose length is longer than 5,000 bps. It is likely that these intronic miRNAs can have their own independently regulated transcription units, which can be regulated by RNA polymerase II (Pol II) or RNA polymerase III (Pol III). It was recently demonstrated that RNA Pol III could transcribe human miRNAs through associated repetitive elements. Since various repetitive elements are often found to be present in the intronic regions, the distribution of intronic miRNAs and their possible transcription regulation are presented. Although the intronic miRNAs and their host genes could be regulated independently, it is possible that the intronic miRNA can still down-regulate its own host protein-coding gene by targeting the untranslated region (UTR) of the host gene. Another biological implication is that intronic miRNAs could play an important role as negative feedback regulators. We propose hypothetical models of such feedback regulation on host protein-coding genes by selecting the transcription factors as miRNA targets or by protein-protein interactions between intronic miRNA host gene product and miRNA target gene products.